Bone marrow derived dendritic cell (BMDCs) generation and lentivirus transduction

Murine bone marrow was flushed from femur and tibiae of C57BL/6 mice, and then passed through 40 μm filter to achieve single cell suspension. After red blood cell lysis, Progenitors were washed and plated with 5 × 10⁶ cells in complete media (RMPI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and streptomycin (P/S), 2 mM L-glutamine, and 1 mM sodium pyruvate) with 20 ng/ml GM-CSF (R&D) and 20 ng/ml IL4 (R&D) for 3 days. On day 4, immature DCs were transduced with pLKO.1-puro empty vector or fascin-shRNA containing lentivirus using 10 μg/ml polybrene (Santa Cruz). After 16 hr incubation, viruses were removed and replaced with fresh complete media supplemented with 20 ng/ml GM-CSF and 20 ng/ml IL4. On day 6, BMDCs were treated with 2 μg/ml puromycin for 2 days and harvested for fascin knockdown verification by western blotting or functional assay on day 8.